RESUMEN
Vacuum infiltration-centrifugation (VIC) is the most reproducible technique for the isolation of apoplast washing fluid (AWF) from leaves, but its effectiveness depends on the infiltration-centrifugation conditions and the anatomical and physiological peculiarities of leaves. This study aimed to elaborate an optimal procedure for AWF isolation from the leaves of Tartary buckwheat grown in in vivo and in vitro conditions and reveal the leaf anatomical and physiological traits that could contribute to the effectiveness of AWF isolation. Here, it was demonstrated that leaves of buckwheat plants grown in vitro could be easier infiltrated, were less sensitive to higher forces of centrifugation (900× g and 1500× g), and produced more AWF yield and apoplastic protein content than in vivo leaves at the same forces of centrifugation (600× g and 900× g). The extensive study of the morphological, anatomical, and ultrastructural characteristics of buckwheat leaves grown in different conditions revealed that in vitro leaves exhibited significant plasticity in a number of interconnected morphological, anatomical, and physiological features, generally driven by high RH and low lighting; some of them, such as the reduced thickness and increased permeability of the cuticle of the epidermal cells, large intercellular spaces, increase in the size of stomata and in the area of stomatal pores, higher stomata index, drop in density, and area of calcium oxalate druses, are beneficial to the effectiveness of VIC. The size of stomata pores, which were almost twice as large in in vitro leaves as those in in vivo ones, was the main factor contributing to the isolation of AWF free of chlorophyll contamination. The opening of stomata pores by artificially created humid conditions reduced damage to the in vivo leaves and improved the VIC of them. For Fagopyrum species, this is the first study to develop a VIC technique for AWF isolation from leaves.
RESUMEN
The major mechanism of gravitropism that is discussed for herbal plants is based on the nonuniform elongation of cells located on the opposite stem sides, occurring in the growing zone of an organ. However, gravitropic response of flax (Linum usitatissimum L.) is well-pronounced in the lower half of developing stem, which has ceased elongation long in advance of plant inclination. We have analyzed the stem curvature region by various approaches of microscopy and found the undescribed earlier significant modifications in primary phloem fibers that have constitutively developed G-layer. In fibers on the pulling stem side, cell portions were widened with formation of "bottlenecks" between them, leading to the "sausage-like" shape of a cell. Lumen diameter in fiber widening increased, while cell wall thickness decreased. Callose was deposited in proximity to bottlenecks and sometimes totally occluded their lumen. Structure of fiber cell wall changed considerably, with formation of breaks between G- and S-layers. Thick fibrillar structures that were revealed in fiber cell wall by light microscopy got oblique orientation instead of parallel to the fiber axis one in control plants. The described changes occurred at various combinations of gravitational and mechanical stimuli. Thus, phloem fibers with constitutively formed gelatinous cell wall, located in nonelongating parts of herbal plant, are involved in gravitropism and may become an important element in general understanding of the gravity effects on plants. We suggest flax phloem fibers as the model system to study the mechanism of plant position correction, including signal perception and transduction.
Asunto(s)
Lino/fisiología , Gravitropismo/fisiología , Floema/fisiología , Pared Celular/metabolismo , Pared Celular/ultraestructura , Lino/crecimiento & desarrollo , Lino/ultraestructura , Glucanos/metabolismo , Floema/anatomía & histología , Floema/citología , Floema/ultraestructura , Tallos de la Planta/fisiología , Tallos de la Planta/ultraestructura , Xilema/fisiología , Xilema/ultraestructuraRESUMEN
The gelatinous type of secondary cell wall is present in tension wood and in phloem fibers of many plants. It is characterized by the absence of xylan and lignin, a high cellulose content and axially orientated microfibrils in the huge S2 layer. In flax phloem fiber, the major non-cellulosic component of such cell walls is tissue-specific galactan, which is tightly bound to cellulose. Ultrastructural analysis of flax fiber revealed that initiation of gelatinous secondary cell wall formation was accompanied by the accumulation of specific Golgi vesicles, which had a characteristic bicolor (dark-light) appearance and were easily distinguishable from vesicles made in different tissues and during the other stages of fiber development. Many of the bicolor vesicles appeared to fuse with each other, forming large vacuoles. The largest observed was 4 mum in diameter. Bicolor vesicles and vacuoles fused with the plasma membrane and spread their content in a characteristic "syringe-like" manner, covering a significant area of periplasm and forming "dark" stripes on the inner wall surface. Both Golgi derivatives and cell wall layers were labeled by LM5 antibody, indicating the presence of tissue- and stage-specific (1-->4)-beta-galactan. We suggest that this specific type of galactan secretion, which allows coverage of a large area of periplasm, is designed to increase the chance of the galactan meeting the cellulose microfibrils while they are still in the process of construction. The membrane fusion machinery of flax fiber must possess special components, which may be crucial for the formation of the gelatinous type cell wall.